Because of the sudden onset of "flu-like" symptoms in the vast majority
of cases, followed by persistent illness and fatigue over several years,
both RNA (retroviruses) and DNA (herpesviruses and enteroviruses) viruses
have been suspected to be implicated in the pathogenesis of CFS. In recent
years, evidence of the association of some viruses with CFS has progressed,
whereas, with some others it has weakened considerably. Thus far, no single
virus has been found to be the causative agent of CFS. Reactivation, however,
of latent virus or viruses could contribute to the symptomatology of CFS
by damaging the immune system either directly or indirectly. In this report
we have provided a comprehensive review of the status of research on viral
agents which have been investigated for their role in the pathogenesis
of CFS.

The evidence implicating a virus in CFS is
specifically based on the sudden onset of "flu-like" symptoms, in the vast
majority of cases, followed by persistent illness and fatigue (1-3). Over
the years, many different RNA and DNA viruses have been suspected of being
associated with CFS, e.g., enterovirus, measles, rubella, influenza, cytomegalovirus
(CMV), Epstein-Barr virus (EBV), Human herpesviruses 6 and 7 (HHV-6, HHV-7),
hepatitis-C, human T-lymphotropic viruses 1 and 2 (HTLV-I, HTLV-II) -like
virus, spuma (foamy) virus and "stealth virus." It has also been suggested
that CFS, also known as chronic mononucleosis-like syndrome, NK cell disease,
Royal Free Disease, post-viral chronic fatigue syndrome and myalgic encephalitis,
may be a neuromuscular disorder, a sequela of an apparent viral illness
(2,4). Although reactivation of some viral agents has been observed in
CFS patients, thus far, no particular virus has been implicated as the
etiological agent in CFS (5). The majority of investigators, and the National
Advisory Council of the American Association for CFS, strongly support
the hypothesis that CFS may be a result of more than one viral agent, and
that the syndrome may be triggered by a variety of organic factors. In
the early years of CFS investigation, Professor Peter O. Behan, of the
University of Glasgow, Scotland, and Professor Mary Ann Fletcher, of the
University of Miami, Florida, proposed that multiple factors are associated
with the pathogenesis of CFS. Of particular interest were the immune disturbances
which are probably the result of virus infection, and which perhaps account
for the clinical features associated with CFS. More recently, Komaroff
et al. (1) view CFS as centrally an immunologic disturbance which allows
reactivation of latent and ineradicable infectious agents, particularly
viruses. The reactivation of such viral agents may only be an epi-phenomenon.
Komaroff, however, believes that it is more likely that, once reactivated,
these viruses contribute to the morbidity of CFS by directly damaging certain
tissues (e.g., pharyngeal mucosa) and indirectly, by eliciting an ongoing
immunologic response. On the other hand, Levy (6) believes that, although
a viral agent has not been found in CFS, an infectious organism may be
the cause of this condition, and may continue to be present in the individual.
Levy proposes an alternative concept which he calls "hit and run," based
on the lack of recovery of an agent. By this hypothesis, a virus might
enter the host, cause immune abnormalities leading to CFS, and then be
eliminated. The immune system may, however, not fully recover. According
to Levy, it appears that the reduction in NK cell function and/or CD8+
T-cell suppression activity (CD8+ lib+ cells) is involved. Thus the hyperactive
immune state resulting from the viral infection, as found in an acute viral
illness, continues in the host. Levy further compares CFS with autoimmune
disease, where autoimmunity could also be caused by an agent no longer
present in the patient, but which has induced a pathogenic course. Thus,
according to these hypotheses, CFS appears to be a disorder of the immune
system caused initially by an infectious agent. This statement is supported
by the fact that frequent immunologic abnormalities are associated with
CFS, e.g., depressed NK cell numbers, impaired NK cell function, abnormal
CD4+ and CDS+ cell numbers and ratio, T-cell cytotoxicity, abnormalities
in the complement cascade, short-term elevation of HLA-DR and CD38+, decreased
lymphocyte response to pokeweed mitogen and phytohemagglutinin (PHA), abnormal
numbers of B-cells, macro-phages and neutrophil function, IgG subclass
deficiencies, skin test allergy and increased expression of ICAM 1 and
LFA-1 in the cell (7-10).

Other than these hypotheses, one must also
consider that one viral agent could activate another virus, which, in turn
may contribute to disease manifestations. Another possibility could be
that there is a direct interaction of two viruses, leading to disease manifestation.
Still another possibility exists that a virus may contribute indirectly
by inducing certain cytokines production by the activated T-cells or macrophages,
e.g., IL-1, IL-2, TNF-alpha, -beta, IL-6 (11-13), These cytokines are known
to give rise to a variety of symptoms when administered to individuals
(fever, myalgia, nausea, anorexia, fatigue, confusion, hypotension). It
is thus conceivable that a virus or combination of several viruses are
involved in the pathogenesis of CFS by causing the immune dysregulation,
and this, in turn, may lead to reactivation of such viral agents. Therefore,
we must investigate the immune system more thoroughly in order to understand
the role of virus in CFS.

In recent years the following RNA and DNA viruses
have been investigated in connection with CFS:

Human herpesviruses, i.e., CMV, EBV, HHV-6
and HHV-7 are ubiquitous agents and their primary infections often result
in acute illness (e.g., infectious mononucleosis, roseola infantum) follow
by viral latency in various cells of the body. Because of their high prevalence
rates of infection in general populations, it is very difficult to prove
their causative roles. Because of their high reactivation rates, however,
significant and increased antibody titers are highly suggestive that they
must play a role in the symptomatology of CFS

EBV

EBV (Human Herpesvirus-4), discovered in 1964,
is a member of the gamma herpesvirus sub-family. It is the etiological
agent heterophile antibody positive infectious mononucleosis and is transmitted
via salivary contact. The oropharynx is the principal site of virus replication
and the virus infects B-lymphocytes. The association of EBV with CFS (2,3,9,14-24)
was based on:

In one study, Ablashi et al. (15) found
that 25% of the sera &am 300 CFS patients, tested for HHV-6 IgG and
EBV-VCA IgG antibody, showed elevated antibodies to both HHV-6 and EBV.
In recent studies by Swanink et al. (25) and Ablashi et al. (26) no evidence
was found of EBV reactivation in selected CFS patients with high titers
of IgG antibody to EBV-VCA and EA. In Swanink's study of 10 CFS patients
and 9 healthy controls, immunological regression of in vitro transformed
peripheral blood mononuclear cells (PBMC) was equally efficient in patients
and controls, whereas, in the study by Ablashi, PBMC from CFS patients
failed in culture to show EB virus over a period of three weeks. This suggested
that, in these patients, both of these herpesviruses were reactivated.
Over the years, evidence of the involvement of EBV in CFS patients is diminishing
(22-26). There may, however, be a subset of CFS patients in whom EBV may
be a major contributing factor to disease manifestation. Finding antibody
to EBV-EA-D suggests that there is reactivation of EBV. It would, therefore,
be important to continue to follow these patients looking for an increase
or decrease of EA-D antibody, and its corelation with symptoms, particularly
after treatment with anti-herpesviral agents, e.g., Acyclovir, Phosphonoformate,
Ampligen, and Kutapressin.

HHV-6 is a member of the herpesviridae and
was originally isolated from patients with AIDS and lymphoproliferative
disorders in 1986 (27). It is the etiologic agent of Roseola infantum,
other febrile illnesses in young children and of non-EBV and non-cytomegalovirus
infectious mononucleosis. HHV-6, like human retroviruses, HIV, HTLV-I and
II, predominantly infects T-lymphocytes, but can also infect other cell
types including fibroblasts, epithelial cells, natural killer cells, rnegacaryocytes,
neural cells, and occasionally B-lymphocytes. HHV-6 can be isolated from
peripheral blood lymphocytes and with difficulty from saliva. Transmission
of HHV-6 is poorly understood (28). There has been considerable interest
in investigating its possible role in CFS. As evidenced by the HHV-6 prevalence
rate of >85% in the U.S. population, most of us have already been infected
with the virus in our first year of life. In most individuals the virus
is latent. It may be, thus, that when HHV-6 is reactivated, or during reinfection,
it may contribute to CFS. Evidence of the involvement of HHV-6 in CFS (1-3,9,15,19,29-36),
compared to that of other human herpesviruses (EBV, CMV, HSV-1 and 2, VZV,
HHV-7), is much stronger. The evidence is based on:

1. elevated IgG antibody ( equal to or more than 3-4 fold);
2. detection of anti-IgM antibody in equal to or less than 50% of patients,
which is a good indication of virus reactivation;
3. detection of HHV-6 antigen expressing cells in the peripheral blood
mononuclear cells of CFS patients by culture techniques; and
4. detection of HHV-6 DNA in lymphocytes of CFS patients by PCR and Southern
blot hybridization (22-23,33,35-36).

The data from at least three different,
independent laboratories outside the U.S. confirmed and extend the findings
of Ablashi et al. (15) and Buchwald et al. (29), and others who have reported
that the HHV-6 reactivation rate in CFS patients is extremely high, compared
to healthy individuals (34,36-37). In a recent study by Yalcin et al. (37),
13 CFS patients and 13 healthy controls from Japan were analyzed for HHV-6
DNA by variant-specific PCR. Seven patients (53.8%) were positive for HHV-6
DNA, while none of the controls was positive. HHV-6 DNA to variant A was
detected in the PBMC of 4 patients, another 3 had HHV-6 DNA to variant
B. These results suggest activated replication of HHV-6 in CFS patients.
Ablashi et al. (26) presented data at the recent Research and Clinical
CFS Conference that 65% of CFS patients' PBMC in culture expressed HHV-6
antigens compared to 25% of healthy controls, when tested under code. Using
an HHV-6 antigen capture assay to core protein gpl16 (38), 54.7% of culture
positive samples were also positive for this protein, compared to only
15% of the healthy controls. Using ELISA to HHV-6 early proteins (P41/38)
the mean IgG antibody in CFS patents was 28.3 +/- 3.0, compared to 8.2
+/- 5.8 in healthy donors. These data using three different assays showed
a high frequency of HHV-6 reactivation in CFS patients. In more recent
findings by Krueger et al. (39) from Germany, 72% of CFS patients exhibiting
significantly elevated IgG antibody to HHV-6 was suggestive of ongoing
HHV-6 infection. These observations were confirmed by cell culture of patients'
PBMC and by antigen capture assay to HHV-6 core protein. By these two assays,
38.6% of patients were positive for virus isolation and core antigen. EBV
replication was not detected in these patients. Since HHV-6 is an inducer
of monocyte/macrophage cytokines IL-l beta and TNF-alfa (40), it may contribute
to CFS in another way. Recently, Lusso et al. (41) showed that HHV-6 can
infect and replicate in subsets of NK (natural killer) cells. Because impaired
NK cell function and a decrease in NK cell numbers in CFS patients is a
consistent finding, it provides evidence that HHV-6 can directly target
and kill NK cells, a potential strategy to suppress natural antiviral immunity
of the host. There have been no reported studies showing how HHV-6 reactivation
contributes to the symptoms of CFS patients, nor has anyone yet reported
finding HHV-6 in NK cells of CFS patients.

Human Herpesvirus-7 (HHV-7)

HHV-7 was isolated in 1990 from a healthy
individual's CD4+ T-lymphocytes after mitogen stimulation (42). HHV-7 was
later, independently reported from the peripheral blood mononuclear cells
of a CFS patient (43). The prevalence rate of HHV-7 is >85-96% in most
of the populations, however, it is approximately 60% in Japan (44). The
primary infection of HHV-7 occurs later than HHV-6. Because of its presence
in CFS patients, the sera of 30 CFS patients and 17 healthy donors provided
by Dr. Komaroff were tested in a blind study for IgG antibody to HHV-7
by IFA. The geometric mean titer of IgG antibody in patients compared to
con trols was 87.1 5 3.9 versus 38.9 + 5.5 (P = 0.078 by two-tailed t test).
The difference between the titers was not statistically signifi cant, suggesting
no elevation of antibody to HHV-7. It did not, however, exclude the association
of HHV-7 in some cases of CFS (45). Secchiero et al. (46) detected high
titer neutralizing antibody to HHV-7 in sera from patients with CFS, compared
to controls, suggesting that HHV-7 in CPS may be related to the immune
dys function. More studies are needed to assess whether HHV-7 is reactivated
in CFS, as is HHV-6.

STEALTH VIRUS FROM A CFS PATIENT

Another DNA virus with related sequences to
CMV, designated the "stealth virus," was reportedly cultured from a 43-year-old
patient by Martin et al. (47). Although viral particles resemble CMV, specific
antisera for CMV, HSV and HHV-6 failed to react with cells infected with
stealth virus. The PBLs from CFS patients after separation of the mononuclear
cells were initially cultured on human foreskin fibro blasts. Cytopathic
effects (CPE) were observed, and CPE was transfer able to subsequent cultures.
The authors claim that they also cultured the virus from the patient during
a 1991 hospital admission. The plasmid 1.5-5-4 from the virus showed partial
homology with only CMV, by PCR. Morphology of the virus revealed enveloped
viral particles, consistent with a herpesvirus. CPE, however, resembled
foamy cell syncytia. The authors also suggested that the virus is present
in CFS patients and is non-inflammatory and neurotropic. Since this is
only one report from one case, the precise association of this virus in
CFS needs further investigation and confirmation by others, as well as
isolation from other CFS patients.

ENTEROVIRUSES IN CFS

Enteroviruses belong to the family Picoma viridae,
and include poliovirus (3 serotypes), Coxsackie A virus (23 serotypes),
Coxsackie B virus (6 serotypes), echoviruses (30 sereotypes) and the newer
enteroviruses (68-71 serotypes). Human hepatitis virus has also been classified
as enterovirus 72. Enteroviruses are small, non-enveloped, icosahedral,
single-stranded RNA viruses with 7.5 kilbase genome. Enteroviruses are
responsible for a variety of illnesses, with approximately 80% of the infections
being asymptomatic. Coxsakie A viruses produce aseptic meningitis, herpangina
and hand, foot and mouth disease. Coxsackie B virus, which has been linked
to CFS, produces myocarditis and pericarditis, generalized disease in new
barns, pneumonia, rashes and common colds. The region of highest homology
between different enteroviruses is a 5'LTR, and most investigators choose
this DNA probe for PCR work.

The majority of work on the role of enteroviruses
in CFS patients has been done in the UK (48-49). The evidence of circulating
anti gen and IgM complexes was found in the majority of CFS patients, with
the virus being isolated in 22% of patients. Archard et al. (48) studied
96 CFS patients using molecular hybridization with entero virus group-specific
probes to RNA isolated from quadriceps muscle biopsies. Only 20 patients
were found positive for enterovi rus RNA, suggesting viral replication.
Gow et al. (49) used PCR to detect enteroviral RNA sequences in muscle
biopsies from 53% of CFS patients and from 15% of controls. Landay et al.
(9) found a higher seroprevalence rate of coxsackie B virus in CFS patients,
however, Miller et al. (50) detected no differences between the prevalence
rates for CFS patients and controls for Coxsackie B by IgM and neutralizing
antibodies. The data presented by Gow et al. (51) at the International
CFS Conference held in Albany, NY, in 1992, on the detection of enterovirus
sequences and virus from patients with post-viral fatigue syndrome did
not find any specific enterovirus type involved in CFS. Contrary to their
earlier studies, however, the present data does not show any significant
differences between patients and controls for the presence of enterovirus
(51).

Swanink et al. (52) reported at the CFS
Research and Clinical Conference that no differences were detected between
CFS patients and controls for enteroviral antibodies by complement fixation
assay and antigen capture ELISA, since they found similar preva lence rates
of VP-1 antigen in sera from patients and controls. Furthermore, enterovirus
was not isolated either by direct culture or culture acid elution in any
of the stool specimens in Swanink's data, therefore supporting the recent
findings of Gow et al. (52), which hypothesize that enteroviruses do not
play any role in the etiology of CFS.

RETROVIRUSES IN CFS

Retroviruses belong to the family Retro viridae
and, as such, they are enveloped, negative-stranded RNA viruses with a
genome size of 9 kilobases. Human retroviruses are relatively unstable
and are quickly inactivated by alcohol, detergents and 0.5% sodium hypo-chlorite
solutions. HTLV-I (Figure 2) has been implicated in T-cell leukemia and
has been linked to chronic neurologic diseases (tropical spastic parapareses).
HTLV-I is poorly contagious and is not easily transmitted by cell-free
body fluids. Transmission can take place, however, through blood cell transfusion,
contaminated needles, sexual contact and from mother to child through breast
milk. Another illness called HTLV-I-associated myelopathy (HAM) has been
observed in all parts of the world.

HTLV-II was first isolated in 1982 from patients
with a T-cell variant of hairy cell leukemia. Very little is known about
the virus. Recent reports suggest that the virus is prevalent in drug users
in New York, New Orleans, England, Milan, Italy, and in other population
centers worldwide. HTLV-I and HTLV-II are closely related and show antibody
cross reactivity. HTLV-II has not yet been linked to any disease. HIV,
although a human retrovirus, is a lentivirus, and a considerable amount
of lentiviruses have been reported from animals (horses, cattle, sheep,
etc.).

Among the retroviruses, two major subgroups
have been studied in CFS, i.e., HTLV-II-like and spuma viruses (29,32,53-60).
First, antibody to HTLV-I and -II were studied by investigators in the
laboratory of Dr. Robert Gallo at the National Cancer Institute, NIH, and
they found no evidence of HTLV-I or -II in sera obtained from CFS patients
from Lake Tahoe. DeFreitas et al. also tested sera from CFS patients from
Lake Tahoe and found no antibody to HTLV-I (55). Ablashi and associates
found two sera positive, one for HTLV-I and the other for HTLV-II when
testing 70 sera provided by Dr. Kornaroff from his CFS patients (unpublished
data). The seroprevalence rate of HTLV-I in the U.S. population is <0.01%;
however, the rate in Native Americans is >20%. The seroprevalence of HTLV-II
is even less, but in IV drug users the rate is between 20-30%. Further
studies on antibodies to HTLV-I were performed by Buchwald et al. (29)
and Levine et al. (32) in CFS patients and controls. None of the patients
or controls exhibited antibody to HTLV-I, nor did Buchwald find evidence
of any human retrovirus using PHA-stimulated peripheral blood mononuclear
cells(29).
DeFreiata et al. (55) reported in April 1991
that HTLV_IIlike viral sequences were detected in the peripheral blood
lymphocytes obtained from adults and children with CFS. Such vial sequences
were not detected in the lymphocytes of healthy individuals. These investigators
did detect the antibody to viral gag proteins, by West ern blot, in the
sera of CFS patients which contained viral sequences. The finding of an
HTLV-II-like virus raised many questions, such as whether the virus is
transmitted sexually and how infectious is this virus. Independent reports
from CDC (58), from Scotland by Gow et al. (59), and Levy (personal communication)
found no evidence of a retrovirus in CFS patients. In fact, no evidence
of animal retroviruses has been found in lymphocytes of CFS patients. At
the International Conference on CFS/ME in 1992, He neine and co-workers
(58) and Gunn and co-workers (60) reported the lack of evidence of a retrovirus
in CFS patients. In Gunn's study, which was later published, samples were
collected from four different centers, with the study performed on coded
samples by Oncore Analytics, Houston, Texas. In the original CARA assay,
the CFS patients and healthy controls were 40% positive for both, and in
the new CARA, 3% of patients were positive compared with 1.5% of controls.
Dr. Gunn's study (60) concluded that the tests performed by Oncore Analytics
did not distinguish between CFS cases and matched controls in a blind study.
Dr. Gunn further stated that the present results, and those reported by
others, did not support the theory of a retroviral etiology in CFS, but
such a possibility still exists. Since the test detected HTLV-II-like sequences
in patients and controls, one may ask whether these sequences may be nonspecific
and cellular rather than viral.

SPUMA VIRUSES

Spuma viruses (Latin term for foamy viruses)
(Figure 3) were first identified in the 1950s as contaminating viral agents
which caused cytopathic effects in cultures of rhesus monkey kidney cells.
The cytopathology of infected cells is characterized by extensive forma
tions of intracellular vacuoles in multinucleated syncytial cells. The
first human spuma virus was identified in 1971. Dr. Martin, at the University
of California, reports finding spuma virus in a significant number of CFS
cases (56). Dr. Levy, in whose laboratory foamy viruses were studied, previously
found no evidence of spuma virus in CPS patients. Flugal et aL (57) also
found no evidence of spuma virus in CFS or in controls. Test on cell cultures
from CFS patients and controls for the cytopathic effects of human spuma
virus conducted by Dr. Martin on coded samples did not differentiate between
patients and controls for the presence of human spuma virus. This data
showed that human spuma virus is not associated with CFS (60).

RETROVIRUS-LIKE PARTICLES

At the CFS Research and Clinical Conference
1994, Diack et al. (61) reported the identification of retrovirus particles
from the pe ripheral blood lymphocytes of 10/34 CPS patients and none of
the controls. The majority of viral particles were similar to the ultra
structure of visna virus (lentivirus) and murine leukemia virus. According
to the authors, virus-like structures were compatible with various maturation
stages of lentivirus. No reverse transcrip tion activity or possible target
cell phenotype was found. Considerable work is needed to prove that these
ultra-structures resembling a retrovirus are not artifacts.

The above review of RNA and DNA viruses
in CFS does not identify any specific virus in the etiology of CFS. It
is most likely that CFS has more than one causative factor. Some of these
may trigger the pathologic changes of CFS, either directly or indirectly,
and may not be required to maintain the syndrome. It can be con cluded
that the end result of viral infection in CFS is the dysregula tion of
the immune system or vice versa. The data reviewed here also show that
it is difficult to prove the role of a herpesvirus in CFS, since most of
us carry them throughout our lives. Some envi ronmental or organic factors,
however, are responsible for the reac tivation of a virus from a latent
state. If the virus is reactivated, as suggested by Komaroff, this may
contribute directly or indirectly to the pathogenesis of CFS, the end result
of which are the abnormali ties of the immune system. The reason for the
immune dysfunction in individuals with CFS is unknown, but the immune system
must be brought back into balance to enable the reversal of the symptoms
associated with this disorder (63). However, individuals with CFS have
two basic changes in immune status, e.g., chronic immune activation and
poor immune cell function with decreased natural killer cell cytotoxic
activity (13). The study of lymphokines in CFS patients holds many promising
leads into the pathophysiology of CFS. It is also evident that longitudinal
follow-up studies are needed to define the role of any virus in the symptomatology
of CFS by correlating the severity of symptoms to viral reactivation. These
studies are also needed to help us understand the underlying mechanism
leading to the pathogenesis. It is also possible that we may not yet have
found the agent which is etiologic, as suggested by Levy.

If we believe that a reactivated virus or viruses
contribute to the symptomatology of CFS, antiviral agents should be recommended
to treat the viral infection and to see if such a treatment would offer
relief in the disease manifestations.

ACKNOWLEDGMENTS

The authors thank Ms. Louise Lane of Advanced Biotechnolo gies Inc.,
Columbia, MD, for excellent secretarial assistance. We would also like
to thank Dr. A. Komaroff of the Harvard School of Medicine, Boston, MA,
Dr. J. Levy of the University of California, San Francisco, CA, and Ms.
Orvalene Prewitt of the National Chronic Fatigue Syndrome and Fibromyalgia
Association, Kansas City, MO for their helpful comments in the preparation
of this article.

Dharam V. Ablashi is affiliated with the Advanced Biotechnologies, Inc.,
Columbia, MD 21046, and is Adjunct Professor of Microbiology, The Georgetown
University School of Medicine, Washington, DC 20007.

Kristine L. Ablashi is affiliated with the Corresponding Office of the
American Association for Chronic Fatigue Syndrome and the International
Institute of Immunopathology, Olney, MD 20832.

Bernhard Kramarsky, John Bernbaum and James E. Whitman, Jr. are affiliated
with the Advanced Biotechnologies, Inc., Columbia, MD 21046.

Gary R. Pearson is affiliated with the Department of Microbiology and
Immunology, The Georgetown University School of Medicine, Washington, DC
20007.